Protein Kinase C-θ and vimentin modulate multiple facets of Regulatory T-cell function.

Abstract

Abstract Regulatory T-cells (Tregs) prevent autoimmune and alloimmune reactions. Augmenting Treg function may enhance Treg therapies for these diseases. Treg-specific inhibition of protein kinase C-theta (PKC-θ) enhances Treg function. However, it is unclear whether PKC-θ inhibition can boost Treg function in systemic inflammatory conditions. In a mouse model of acute GVHD, we found that Tregs treated with the PKC-θ inhibitor AEB071 reduced GVHD mortality and severity significantly better than DMSO treated Tregs. Compared to DMSO, AEB071 treated Tregs significantly reduced conventional T-cells (Tcon) proliferation on D4 after transplant. Multi-photon microscopy showed that AEB071 treated Tregs significantly increased Tcon velocity and displacement compared to DMSO. Mechanistically, AEB071 augments expression of Neuropilin-1 (Nrp1) and Lymphocyte activation gene 3 (Lag3). Antibody blockade of Nrp1 and Lag3 in transwell suppression assays reduced the effect of AEB071 on Treg function. PKC-θ inhibition also reduces phosphorylation of mTORC2 targets FoxO3a and Akt phospho-site S473, but not mTORC1 targets S6, 4E-BP1 or Akt phospho-site T308. Compared to DMSO, AEB071 treatment significantly increased fatty acid uptake and oxygen consumption rate (OCR). Phosphoproteomic analysis identified a significant alteration in the interaction between PKC-θ and the intermediate filament vimentin after AEB071 treatment, which was confirmed by confocal. Vimentin siRNA treatment also significantly reduces PKC-θ/vimentin interaction, increases Treg function, Nrp1 expression and OCR. In summary, PKC-θ and vimentin modulate multiple aspects of Treg function, and altering these molecules may enhance the efficacy of Treg therapeutics.